Purpose: Osteoarthritis (OA) is a chronic and debilitating disease of articulating joints. Micro-RNAs (miRNA) are endogenous, short ∼22-nucleotides non-coding single stranded RNAs that act as a regulator of gene expression. In order to understand the role of miRNAs in OA pathogenesis we employed deep sequencing technology to comprehensively profile the miRNA population in OA chondrocytes.
Methods: OA Chondrocytes were prepared from human cartilage samples obtained from OA patients at the time of knee arthroplasty and were treated with IL-1β (2ng/ml) for 1, 12, 24 h. Total RNA from cartilage was obtained using Trizol and from chondrocytes using miRNeasy Mini kit (Qiagen). Small RNA library was prepared using TrueSeq Small RNA Library Preparation Kit (Illumina). RNA purity, integrity and library integrity was verified using Agilent 2100 Bioanalyzer. Cluster generation and sequencing was performed on MiSeq (Illumina). The small RNA-Seq reads were aligned to genomic reference (hg19) and subsequently miRNAs were annotated to generate the miRNA abundance profile and novel miRNA sequences from NGS data using Strand NGS software. Data was normalized followed by differential expression analyses in control and treated samples using Strand NGS software. Differentially regulated miRNAs were used to construct the signaling pathways that were enriched by the treatment using DIANA-mirPath open web tool. To detect the novel miRNAs, cDNA was synthesized using miScript II RT kit employing miScript HiSpec buffer which enables to quantify mature miRNA using individual miScript Primer Assay.
Results: We obtained a total of ∼20 million reads and more than 95% reads pass the quality filter. We detected 1548 differentially expressed miRNAs in our sequencing reads. miRNAs with less than 5 sequencing reads were excluded from the analyses which left 511 differentially expressed miRNAs. In OA chondrocytes, expression of 10 miRNAs (miR-100, 26a, 22, 148a, 27b,125b, 99b, 21, let-7a and 7b) on average constitute ∼70% of all miRNAs expression across the samples. We identified 46 miRNAs that were upregulated and 41 miRNAs that were downregulated compared to untreated control sample at all the time points analyzed (1.5 fold cut-off). Highest upregulated miRNA was miR-146a (30 fold) which has been implicated in OA pathogenesis and most downregulated miRNA was miR-452 (5 fold). Fifteen miRNAs were identified as potential novel miRNAs by the Strand NGS software. Differential expression of novel miRNA-N103 and N-105 were confirmed in IL-1β stimulated chondrocytes by miScript PCR assay. Interestingly expression of miRNA-N103 and N105 was significantly reduced in damaged cartilage compared to the smooth cartilage. DIANA mirPath analysis of 60 modulated miRNAs identified MAPK signaling, PI3K-AKT signaling, focal adhesion and regulation of actin cytoskeleton as topmost KEGG pathway.
Conclusions: Deep sequencing revealed the expression of several novel miRNAs in OA chondrocytes. Novel miRNA-103 and N105 showed a differential pattern of expression in smooth and damaged OA cartilage. These newly identified miRNAs could have novel and hitherto unexplored functions in OA pathogenesis.
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